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Recent documents in Sluder Laben-usThu, 02 Jul 2015 11:36:05 PDT3600Microscope basicshttp://escholarship.umassmed.edu/sluder/15
http://escholarship.umassmed.edu/sluder/15Wed, 11 Jun 2014 07:05:22 PDT
This chapter provides information on how microscopes work and discusses some of the microscope issues to be considered in using a video camera on the microscope. There are two types of microscopes in use today for research in cell biology-the older finite tube-length (typically 160mm mechanical tube length) microscopes and the infinity optics microscopes that are now produced. The objective lens forms a magnified, real image of the specimen at a specific distance from the objective known as the intermediate image plane. All objectives are designed to be used with the specimen at a defined distance from the front lens element of the objective (the working distance) so that the image formed is located at a specific location in the microscope. Infinity optics microscopes differ from the finite tube-length microscopes in that the objectives are designed to project the image of the specimen to infinity and do not, on their own, form a real image of the specimen. Three types of objectives are in common use today-plan achromats, plan apochromats, and plan fluorite lenses. The concept of mounting video cameras on the microscope is also presented in the chapter.
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Greenfield Sluder et al.Microscopy, FluorescenceMicroscopy, VideoOptical PhenomenaVideo RecordingPractical aspects of adjusting digital camerashttp://escholarship.umassmed.edu/sluder/14
http://escholarship.umassmed.edu/sluder/14Wed, 11 Jun 2014 07:05:20 PDT
This chapter introduces the adjustment of digital camera settings using the tools found within image acquisition software and discusses measuring gray-level information such as (1) the histogram, (2) line scan, and (3) other strategies. The pixel values in an image can be measured within many image capture software programs in two ways. The first is a histogram of pixel gray values and the second is a line-scan plot across a selectable axis of the image. Understanding how to evaluate the information presented by these tools is critical to properly adjusting the camera to maximize the image contrast without losing grayscale information. This chapter discusses the 0-255 grayscale resolution of an 8-bit camera; however, the concepts are the same for cameras of any bit depth. This chapter also describes camera settings, such as exposure time, offset, and gain, and the steps for contrast stretching such as setting the exposure time, adjusting offset and gain, and camera versus image display controls.
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Joshua J. Nordberg et al.AnimalsCells, CulturedHumansImage Processing, Computer-AssistedMicroscopyPhotography*SoftwarePreface. Digital microscopyhttp://escholarship.umassmed.edu/sluder/13
http://escholarship.umassmed.edu/sluder/13Wed, 11 Jun 2014 07:05:19 PDTGreenfield Sluder et al.Image Processing, Computer-AssistedMicroscopy, ConfocalMicroscopy, FluorescenceMicroscopy, VideoSingle-Cell AnalysisCentriole engagement: it's not just cohesin any morehttp://escholarship.umassmed.edu/sluder/12
http://escholarship.umassmed.edu/sluder/12Wed, 11 Jun 2014 07:05:18 PDT
The belief that cohesin complexes link mother to daughter centrioles has received substantial experimental support. New studies challenge the primacy of cohesin in centriole engagement and provide a more nuanced view into the mechanisms for centriole disengagement in anaphase.
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Greenfield SluderAnimalsCaenorhabditis elegansCentriolesCyclin-Dependent KinasesDrosophilaDrosophila ProteinsEmbryo, NonmammalianLink Between DNA Damage and Centriole Disengagement/Reduplication in Untransformed Human Cellshttp://escholarship.umassmed.edu/sluder/11
http://escholarship.umassmed.edu/sluder/11Wed, 11 Jun 2014 07:05:17 PDT
The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 hours, though 14% of the cells eventually go through mitosis in that time. By 72 hours after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with gamma-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53 dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. J. Cell. Physiol. (c) 2014 Wiley Periodicals, Inc.
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Stephen Douthwright et al.Repeated cleavage failure does not establish centrosome amplification in untransformed human cellshttp://escholarship.umassmed.edu/sluder/10
http://escholarship.umassmed.edu/sluder/10Wed, 25 Apr 2012 10:04:51 PDT
We tested whether cleavage failure as a transient event establishes an incidence of centrosome amplification in cell populations. Five rounds of approximately 30% cytochalasin-induced cleavage failure in untransformed human cell cultures did not establish centrosome amplification in the short or long terms. The progeny of binucleate cells progressively dropped out of the cell cycle and expressed p53/p21, and none divided a fourth time. We also tested whether cleavage failure established centrosome amplification in transformed cell populations. Tetraploid HCT116 p53(-/-) cells eventually all failed cleavage repeatedly and ceased proliferating. HeLa cells all died or arrested within four cell cycles. Chinese hamster ovary cells proliferated after cleavage failure, but five rounds of induced cleavage failure produced a modest increase in the incidence of centrosome amplification in the short term, which did not rise with more cycles of cleavage failure. This incidence dropped to close to control values in the long term despite a 2-6% rate of spontaneous cleavage failure in the progeny of tetraploid cells.
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Anna Krzywicka-Racka et al.AnimalsCHO CellsCell ProliferationCells, CulturedCentrosomeCricetinaeCricetulusHeLa CellsHumansImmunohistochemistryTumor Suppressor Protein p53The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturationhttp://escholarship.umassmed.edu/sluder/9
http://escholarship.umassmed.edu/sluder/9Thu, 24 Mar 2011 07:40:19 PDT
It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547-1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous "precentrioles" become morphologically recognizable centrioles before mitosis. De novo-assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo-formed centrioles do not mature if they are assembled in S phase-arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.
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Sabrina La Terra et al.Cell CycleCentriolesGenes, ReporterHela CellsHumansS PhaseTwo-way traffic: centrosomes and the cell cyclehttp://escholarship.umassmed.edu/sluder/8
http://escholarship.umassmed.edu/sluder/8Thu, 24 Mar 2011 07:40:18 PDT
The well recognized activities of the mammalian centrosome--microtubule nucleation, duplication, and organization of the primary cilium--are under the control of the cell cycle. However, the centrosome is more than just a follower of the cell cycle; it can also be essential for the cell to transit G1 and enter S phase. How the centrosome influences G1 progression is a mystery.
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Greenfield SluderAnimalsCell CycleCentrosomeMicrosurgerySignal TransductionSpindle pole fragmentation due to proteasome inhibitionhttp://escholarship.umassmed.edu/sluder/7
http://escholarship.umassmed.edu/sluder/7Thu, 24 Mar 2011 07:40:17 PDT
During interphase, the centrosome concentrates cell stress response molecules, including chaperones and proteasomes, into a proteolytic center. However, whether the centrosome functions as proteolytic center during mitosis is not known. In this study, cultured mammalian cells were treated with the proteasome inhibitor MG 132 and spindle morphology in mitotic cells was characterized in order to address this issue. Proteasome inhibition during mitosis leads to the formation of additional asters that cause the assembly of multipolar spindles. The cause of this phenomenon was investigated by inhibiting microtubule-based transport and protein synthesis. These experimental conditions prevented the formation of supernumerary asters during mitosis. In addition, the expression of dsRed without proteasome inhibition led to the fragmentation of spindle poles. These experiments showed that the formation of extra asters depends on intact microtubule-based transport and protein synthesis. These results suggest that formation of supernumerary asters is due to excessive accumulation of proteins at the spindle poles and consequently fragmentation of the centrosome. Together, this leads to the conclusion that the centrosome functions as proteolytic center during mitosis and proteolytic activity at the spindle poles is necessary for maintaining spindle pole integrity.
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Anka G. Ehrhardt et al.AnimalsAntigens, NuclearBiological TransportCell LineCentrosomeCricetinaeDyneinsHumansInterphaseLeupeptinsMiceMicrotubulesMitosisMitotic Spindle ApparatusNuclear Matrix-Associated ProteinsNuclear ProteinsProteasome Endopeptidase ComplexProtein BindingTranscription, GeneticTubulinUbiquitinWorking with classic videohttp://escholarship.umassmed.edu/sluder/6
http://escholarship.umassmed.edu/sluder/6Thu, 24 Mar 2011 07:40:15 PDT
Discusses the practical aspects of adjusting the video camera and the monitor to prevent the loss of specimen image gray-level information in a video microscopy imaging system.
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Edward H. Hinchcliffe et al.Image EnhancementImage Processing, Computer-AssistedMicroscopy, VideoSignal Processing, Computer-AssistedSpecimen HandlingA sealed preparation for long-term observations of cultured cells: details of support slide constructionhttp://escholarship.umassmed.edu/sluder/5
http://escholarship.umassmed.edu/sluder/5Thu, 24 Mar 2011 07:40:14 PDTGreenfield Sluder et al.Cells, CulturedEquipment and SuppliesLaboratory Techniques and ProceduresCentriole biogenesis: a tale of two pathwayshttp://escholarship.umassmed.edu/sluder/4
http://escholarship.umassmed.edu/sluder/4Thu, 24 Mar 2011 07:40:13 PDT
Two recent studies in Drosophila demonstrate that overexpression of proteins required for centriole duplication can not only induce centriole over-duplication in cells containing centrioles, but can also drive de novo centriole assembly in unfertilized eggs that initially lack centrioles. These studies offer a new perspective on the mechanisms that control centriole duplication.
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Jadranka Loncarek et al.AnimalsCell CycleCell Cycle ProteinsCells, CulturedCentriolesDrosophilaEmbryo, NonmammalianFemaleHumansModels, BiologicalOvumCyclin E in centrosome duplication and reduplication in sea urchin zygoteshttp://escholarship.umassmed.edu/sluder/3
http://escholarship.umassmed.edu/sluder/3Thu, 24 Mar 2011 07:40:12 PDT
When protein synthesis is completely blocked from before fertilization, the sea urchin zygote arrests in first S phase and the paternal centrosome reduplicates multiple times. However, when protein synthesis is blocked starting in prophase of first mitosis, the zygote divides and the blastomeres arrest in a G1-like state. The centrosome inherited from this mitosis duplicates only once in each blastomere for reasons that are not understood. The late G1 rise in cyclin E/cdk2 kinase activity initiates centrosome duplication in mammalian cells and its activity is needed for centrosome duplication in Xenopus egg extracts. Since the half-time for cyclin E turnover is normally approximately 1 h in sea urchin zygotes, the different behaviors of centrosomes during G1 and S phase arrests could be due to differential losses of cyclin E and its associated kinase activities at these two arrest points. To better understand the mechanisms that limit centrosome duplication, we characterize the levels of cyclin E and its associated kinase activity at the S phase and G1 arrest points. We first demonstrate that cyclin E/cdk2 kinase activity is required for centrosome duplication and reduplication in sea urchin zygotes. Next we find that cyclin E levels and cyclin E/cdk2 kinase activities are both constitutively and equivalently elevated during both the S phase and G1 arrests. This indicates that centrosome duplication during the G1 arrest is limited by a block to reduplication under conditions permissive for duplication. The cytoplasmic conditions of S phase, however, abrogate this block to reduplication.
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Bradley J. Schnackenberg et al.AnimalsCell NucleusCentrosomeCyclin ECyclin-Dependent Kinase 2G1 PhasePurinesS PhaseSea UrchinsZygoteProlonged prometaphase blocks daughter cell proliferation despite normal completion of mitosishttp://escholarship.umassmed.edu/sluder/2
http://escholarship.umassmed.edu/sluder/2Thu, 24 Mar 2011 07:40:10 PDT
The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However, some defects are not detected by this checkpoint. With low concentrations of microtubule-targeting agents, the checkpoint eventually becomes satisfied, though the spindles may be short and/or multipolar [3, 4] and the fidelity of chromosome distribution and cleavage completion are compromised. In real life, environmental toxins, radiation, or chemotherapeutic agents may lead to completed but inaccurate mitoses. It has been assumed that once the checkpoint is satisfied and cells divide, the daughter cells would proliferate regardless of prometaphase duration. However, when continuously exposed to microtubule inhibitors, untransformed cells eventually slip out of mitosis after 12-48 hr and arrest in G1 [5-8] (see also [9]). Interestingly, transient but prolonged treatments with nocodazole allow completion of mitosis, but the daughter cells arrest in interphase [10, 11] (see also [9, 12]). Here we characterize the relationship between prometaphase duration and the proliferative capacity of daughter cells. Our results reveal the existence of a mechanism that senses prometaphase duration; if prometaphase lasts >1.5 hr, this mechanism triggers a durable p38- and p53-dependent G1 arrest of the daughter cells despite normal division of their mothers.
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Yumi Uetake et al.Antineoplastic AgentsCell Line*Cell ProliferationHumansLeupeptinsMitosisNocodazolePrometaphaseCentriole duplication: analogue control in a digital agehttp://escholarship.umassmed.edu/sluder/1
http://escholarship.umassmed.edu/sluder/1Thu, 24 Mar 2011 07:40:09 PDT
In preparation for mitosis, the centrosome doubles once and only once to provide the two poles of the mitotic spindle. The presence of more than two centrosomes increases the chances that mitosis will be multipolar, and chromosomes will be distributed unequally. Since the number of mother-daughter centriole pairs determines the number of centrosomes, it is important that only one daughter centriole is assembled at, but slightly separated from, the proximal end of each mother centriole. This numerical and spatial specificity has led to the belief that a 'template' on the mother centriole provides a unique site for procentriole assembly. We review observations that are leading to the demise of this intuitively attractive idea. In its place, we are left with the notion that pericentriolar material at the wall of the mother centriole provides a local environment that promotes the assembly of a macromolecular complex that seeds the daughter centriole. Even though the system normally behaves in a digital fashion to go from zero to just one daughter centriole per mother, this behaviour appears to be based in the precise analogue control of multiple proteins, their activities, and the structure provided by the mother centriole.
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Greenfield Sluder et al.AnimalsCell Physiological PhenomenaCentriolesCentrosomeHumansMitosisMitotic Spindle ApparatusModels, Biological